Nonsense-mediated mRNA decay (NMD) in mammalian cells can be viewed as (i) a quality control mechanism that degrades abnormal mRNAs so as to eliminate the production of potentially deleterious truncated proteins and (ii) a regulatory mechanism that targets normal mRNAs so as to allow for proper levels of gene expression. We have shown that NMD generally degrades mRNAs that terminatetranslation more than 50-55 nucleotides upstream of a splicing-generated exon-exon junction. During the past funding period, we have demonstrated that the role of the exon-exon junction reflects the splicing-dependent deposition of an exon-junction complex (EJC) of proteins, which recruits Upf factors that elicit NMD when translation terminates sufficiently upstream. Furthermore, we have shown that Upfl is phosphorylated by the PIK-related protein kinase Smg1 and dephosphorylated in reactions that depend on at least two of three Smg5/7 factors, which were renamed Smg5, Smg6 and Smg7 once sequences for the C. elegans orthologs became available. This application aims to extend these and related studies. Aim 1 is to continue to examine Upf1 function, including its role in a new mRNA decay pathway that we have uncovered: Staufenl- mediated mRNA decay (SMD). Aim 2 is to define and characterize what we have called mRNA "failsafe" sequences, which can mediate NMD in place of the 3'-most exon-exon junction (i.e., when the 3'-most intron has been deleted). Notably, data indicate that a failsafe sequence elicits NMD only if the mRNA derives from pre-mRNA that harbors at least one intron. Aim 3 is to study the structure of mRNP that is targeted for NMD in S. cerevisiae. To date, we have demonstrated that NMD targets both Cbclp-bound and elF4E- bound mRNAs. The proposed experiments logically extend our long-time studies, and they should lend important insight into aspects of SMD and NMD that have yet to be understood.